LO-CD2a ANTIBODY AND USES THEREOF FOR INHIBITING T-CELL ACTIVATION AND PROLIFERATION

Abstract: The present invention relates to an LO-CD2a antibody and methods of using such antibodies or molecules that bind to a portion of the same epitope (or a portion thereof) to prevent and inhibit an immune response in human patients, preferably, where the immune response is mediated by the activation and proliferation of T-cells or natural killer cells. The administration of an effective amou nt of the LO-CD2a antibody to a human patient will prevent or inhibit graft rejection, graft versus host disease or autoimmune disease.

Description: LO-cca* jurnioDT jun> usn uor FOR XMKIBXSXiα T CI L ACTIVATIO-f AID PROLIFϊl tlOβ This Application is a continuation-in-part of U.S. Patent Application Serial Ho. 08/119,032, filed September 9, 1993, now pending, which ia a oontinuation-in-part of U.S. Patent Application Serial No. 01/027,009 filed March 5, 1993, now pending. This invention relates to an antibody (or fragment or derivative thereof) and preferably, to an antibody (or tragnant or derivative thereof) which bindi to huαan lymphocytes. More particularly, thie invention relates to preventing and/or inhibiting on-going immune responses in a patient through the administration of such antibody (or fragment or derivative thereof) to a patient. Preferably, this invention relates to preventing or inhibiting T cell activation and proliferation through the administration of eueh antibody or fragment or derivative thereof to a patien . The prior art has disclosed the possibility of using antibodies to CD2 antigen for inhibiting graft rejection, In general, the prior art discloses the use of antibodies which bind to CD2 antigens as being possibly uaeful or inhibiting graft RECTIFIEDSHEET(RULE91) ISA/EP rejectlon., see, Qrtho pharmaceutical Corp,, U.S. Pat. No*. 4,364,973; 4,614,720; 4,515,893; 4,743,681; and 4,798,906. Such antibodies have not been Xnown to be useful In inhibiting graft rejection in human patients or in animal* , λs exemplified in the following references, J. V. Giorgi, et al., TimtφioanttDreeelve eff ct and H nunoyenicit pf OKTllλ Monoclonal Antibody in Mon ev Alinprn f wH.piβntn. Transplantation Proceedings Vol. xv Mo. 1, March 19B3, and P. J. Thurlov, et al., A wo ft onϋi ι«»ι-Pn*«.«N.-ff__»ιι snt_ hfMhr. Transplantation, Vol. 36, Mo. 3, Pg. 293-290. Detailed P-»«crJBtlon nf f.ht% Flgiif FIGURE 1 Two color staining of peripheral blood mononuelear eella (PBMC) with biotinylated O-CD2a and eu-5bPE. "■ * " For this staining, the following parameter* were followed: ΠJ- LOO) gmu> LOCATIOSΠ i7.ιs,9 TOTAL - 5000 GATED - 1290 CWAΏ fiUBSft U2&HQ * T Q T^ X KUUf T MBΛW lUL 299 23. IS 3.9β 11.41 284.69 IUR 851 65.97 17.02 32.70 630.65 3 135 10.47 2.70 4.01 3.31 4 R 5 0. 39 0.10 25. 11 6.54 FIGURE 2 Hunan PBKC were stained with L0-CD2a-FlTC and then a) stained with TU-?E<Coulter antibody to CD2) conjugated with phyeoerythrin <PE> or b) Leu-SBHPB (Becton Dickinson antibody to CD2> conjugated to phyeoerythrin (PH). In neither case vaa a aining by the second antibody altered by pretreatnent with LO~ CD2a. RECTIFIECTSHEET(RULE91) ISAEP FISURES 3a and 3b Effects of L0-CD2a on membrane markers. PBKC at 2x10* cells/ml were cultured in the absence (solid lines) or In rhe presence (bro en lines) of LQ-CD2* <200 αg/ l). At the tinea indicated in the figures, cells were harvested and treated for cytofluoromβtrlc analysis, a) and b) ; PBMC were labeled with anti-CD3 <l-.eu-4a-FrτC) f anti-CD* (T4-RD) mAbβ, anti-class II antigens (LQ-DRβ-FITC) or βnti-CDβ (T8-RD) πtonαeolnal antibodies <nλbβ). Negative controls for commercial mou»« nϋfcbs vere aliquots of the aame cells stained with riTC or Λhodβsiine-labeled mouss igGfl. Negative controls for rat nλbs were cells incubated with normal rat serum followed by a FITC-labeled mouse anti-rat nλb (KARK-FITC) , Results are expressed as percentage positive calls* Fisiπuss 4a and 4b effects of LO-CD2a on membrane markers and human blood lymphocyte culture with and without addition of LO-CD2a. Lymphocyte Culture* at 1x10* oellβ al were labeled with {a} anti- CD2 (Lβu-5b-FϊTC), aati-C04 (T4-RD1) π*b, or anti-CD β (TB-RD) πAb at tines indicated; <b) mixed lymphocyte cultures at 1*10* cells per ml of each donor were stained with βnti-CD2 ttλb Leu-Sb (FITC- labeled). Negative controls for commercial mouse mJLbs were aliquotβ of the aame cells stained with FITC or Rhodamine-labeled mouse IgGβ. Negative centrele for rat mAbβ were cells incubated with normal rat serum followed by a FITC-labeled nouse anti-rat mλb (MARK-FI C) . Results are expreesad «a percentage positive cells. FIGURES 5a and 5b Bffeetβ of LO-CD2a and βu-Sb on CD2 expression. Human »MC were inβubeted with a) O~CD2» (300 ng ml ) or b) Lau-5b (dialyzed againat PBS, diluted 1:2) for the time* indicated and a) etained for expression of 2 < eu-Sb-FXTC and Tll-RDl) and RECTIFIED SHEET (RULE 91) JSA/EP -4- for bi din of O-CD2β (Mar.-3-FiTC) or b) CD2 <LO-CD2a-?I7C, Tli- Dl) and for binding of Lβu-5b Goat ar.ti-jnouβf* (GAH-FITC). FIGURE 6 Effects of 0-CD2a on MLR. a) inhibition at MLR in nixed lymphocyte oultυres incubated for 6 days in the presence of increasing concentrations of L0-CO2a added at time 0. Culture* ware harvested &t day 6; b) inhibition of MLR in mixed lymphocyte cultures incubated with different concentrations of LO~CD2a added at time C Cultures were harvested at 24 . intervals; c) *H- Thyioidine (Η-T) incorporation (cpm) by mixed lymphocyte cultures in the absence (solid line) or in the presence (broken line) of Lύ-CD2a (200 ng/ml); d) inhibition of MLR by O-C 2β (200 ng/ml) added at different times after the start of incubation. Cultures vere harvested at day β. All cultures vere made ia triplicate (1x10* cells of each donor/ml) in a final volume of 2Co yl/vell. H-Thymidine wee added β h before harvesting cultures. Results in c) are shown as cpm x io J incorporated per well harvested at the time indicated. Results in a)* b) and d) are expressed as percentage inhibition of MLR of triplicate cultures (aiean ± 8.D.;, as compared to control cultures (without LO-CD2a). rzo uE 7 Peripheral blood ononuclear calls vere cultured in mixed lymphocyte cultures with or without the addition of 200 ng/ml of O-CD3e. At the indicated tines cells were removed and analyzed toy flow cytometry after staining with antibody to CD2 ( eu5b-riTC). Blast cells ware gated by forward and side scatter and the expression of the indicated markers quantified on the blast cells. RECTIFIEDSHEET(RULE91) ISA/EP -3- FI(3UR£ 6 Effects of LO-CDSa on blast: cells- Human lymphoeytaβ were cultured with and without the addition o£ L0-CD2a (200 ng/ml). At the indicated times cells were removed and stained for CD3 (Leu 4e-FXTC), CD4 (T4-RD1) CD8 (TB-RD1) or CD25 (LO- TACT-I-FITC). The resting lymphocytes were identified by differential gating for sice and granularity and the results are expreaaed as the per cent of total resting lymphocytes staining with the indicated antibody. FIGURE B Effects of O-CD2a on mitogen-βtiπtulated lymphocytes. PBKC were cultured for 96 h in the absence (hatched bars) or in the presence (solid bars) of O T3 (100 ng/ml) , Con-λ (10 μg/pd) and PHA (1 μg/ml ) . In parallel cultures, O-CB2a (200 ng/jnl) was added 1 after mito βnβ (gray bars) or 1 h before mitogens (blank bare), βquareβ bars represent cultures performed in the presence of L0-CD2a alone. Cultures (in triplicate) were pulse- labeled with *H-Thymidine during the last fl of incubation. 7IGURBS 10 ø end 10b Inhibition of NK activity by incubation of the effector and target calls ( SI CR labeled K562 cells) in presence of 0-CD2a. Three concentration* of LO-CD2a bave been testedt 5 μg/nl, 1 μ.g/ml, 0.5 μg/ml. Effector cells were peripheral blood lyaaphocyte β of HK activity is expressed as percent lysia of labeled target cells. Two normal βubjecta were tested at 3 ι/τ ratios: 200/1, loo/l, SO/1. ri UKi i Iffects of LO-caaa on mitogβn-driven activation of FKBC . PMBCs from two donors were cultured for 96h in the presence of 0XT3 ( 100 ng/ml) , CON-λ ( 10 g/ml ) and FKA ( 1 μg/ml ) . In parallel cultures , LO-CD2a ( 200 nς nl ) was added at Day 0 ( Qh) RECTIFIED SHEET (RULE 91) ISA/EP -6- after the initiation of the cultur , Bay 1 (24h), or Day 2 (4βh). The graph depicts the percentage inhibition by L0-CD2β of the Λitogen-induc β d proliferation in each donor. FIGURE 12 Total lymphocytes per μl of peripheral blood of a cynomolgus monkey receiving 20 jr.g/day of O-CD2a for 10 days (days o-*). FIGURE 13 PSMC cells from the cynomolgus monkey receiving L0-CD2a at 20 mg/day for 10 days (day 0 to 9) were stained with monoclonal antibodies to CD2 (Lβu-5b), CD4 ( βu3a), CD8 (Leu 2a), Natural Killer cells (CD8 and coiifa), and B calls (antl-IgH) on the days indicated and analyzed by flow cytoroetry. Results are presented as the percentage ot the total number of ataining cells per microliter of blood. ΠSURS KK activity of a eynoroolguβ monkey receiving 20 mg/day of LO-GDSa for 10 days (day 0-9). NX activity was assayed on days 11 and 32 and preeented Λ lysis at E/T of 25/1, 50/1 and 100/1. FIGURE 15 βeru concentration of O-CD-Sa of a cyno olguβ on sy receiving the antibody at 20 mg/day for 10 days (day 0-9). The monoclonal antibody was measured by E&ΣSλ as described in the ex and expressed in μg/ml. riGuxε iβ Development of igG antibody to O-CD2*. in a cynomolgus monkey receiving 20 stg/day of LO-CD2* for 10 days (days 0-9). The antibody to the monoclonal antibody was measured in serial dilutions of serum drawn on the indicated days by the sandwich RECTIFIED SHEET (RULE 91) SA/EP 94/20619 -7- E I8A described in the text and ii expressed as the optical density at 492 nm. FIGURES 17a and 17b I eet of O-CD2a on baboon lymphocytes. a} On the days Indicated blood was obtained from the baboon and cells ware stained with the anti-CD2 antibodies Tll- RDl and Leu-3b-FITC, LO-CD2a and MARM-riTC a mouse anti-rat kalpa lb antibody coupled to fluorescein to detect bound L0-CD2a. b) Serum samplea taken on the indio ted days were evaluated for levels of O-CD2* by ELIIA. FIGURES 18a and 18b t fβct of L0~co2a on baboon lymphocytes. On t e indicated days blood was taken and the cells stained to detect bound 0-CD2α with KλXKS-Pl C and MλlUtfb-a- biotin (a mouse monoclonal anti-rat XgG2b antibody coupled to blotin) detected with PE-coupled streptavidin) . a) No pretreataent of cells; b) incubation with 2.5 μg/al O-CD2a prior to staining to detect any sites unoccupied by circulating antibody. FIGURE 9 Bffect Of O~CD2a on baboon lymphocytes. Oh the indicated days blood samples were taken and stained with T4-RD1 (CD4); T6-H 1 (CD8) or WAK3- ITC (bound LO* C02a). FXCUX820 Leukocytes, lymphocytes and creatlnine in patient II treated with AG than O-CD2e for allograft rejection. FIGURE 22 Serum levels of LO-CD2a during and following treatment in patient #1. RECTIFIED SHEET (RULE 91) JSA/EP W -e- FXGURE 22 Crβatinina in patient 2 prior to, during end following treatment with LO-CD2a. FIGURE 23 Leukocyte and lymphocyte coun a in patient #2 prior to, during and following treatment with LO~CD2a. FIGURE 24 Serum levela of LO-CD2e in patient #2, drawn just prior to and 2.3 hours after each injection. FIGURE 25 Leukocyte count, lyxphoeyta count and serum creatlnine level in patient #3 receiving L0-CD2a for rejection of renal allograft. FIGURE 26 Dual color staining with LO-CD2a and <2) Leuβb, <3) Leu 4<CD3), (4) Leu3s(CD4} r (5) Leu2b<CD8) and (6) Leull <aati-CD16) a marker for NK cells. LO-CD2a binding was detected with goat anti-rat I6-FITC. The upper eat ( l-t) of two color histograms shows the double staining. The low sat (7-12) shows single staining with each antibody. FIGURE 27 Two color staining of human PBL with a rat iaotype control for L0-CD2a (Pharmingen, purified rat rgG2b, kappa) or LO~CD2a and phycoβrthyrin conjugated antibodies to CD4 (c,d>, Cd β <o,f) r CDl6(g h) / CDi»(i,j> and CD2 (k,l). LQ-CD2a and the isotype control ware detected with FITC conjugated affinity purified F(sb')2 anti-rat irwnunoglobulin (Southern Biotechnology) . The antibodies to the CD antigens were all phyeoerythrin conjugated antibodies obtained from Beeton- Diokineon [CD4toeu3a>, cr>8(Leu2a), CDl β (i-βu-Ub), CDlø(Leu 12) RECTIFIED SHEET (RULE 91) ISA/EP end CD2 (LβuSb)]. In each case staining with the isotype control is shown in the first histogram and the LO-CD2a in the ββcond hiβtogrβm. Histogram a shows the pattern with the isotype control and b with iΛ-CD2a. PXGURE 2d Cytofluorograph analysis of the staining of COS cell* tranβfectβd with vild-typβ CD2. The left panels show the histograms of staining of a COS cell tranβfectβd with the control vector, not containing CD2; the right set of panelβ staining of a COS cell transiently transfected with a vector containing the entire CD2 molecule, in each aet the top histogram shows the staining with murine 632 {antibody to Class 1, known to be expressed by COS cells) and 76-2-11 (an isotype control for the murine W632); the middle panel shows staining with βuSb (anti- CD2 from Becton Dickinson) and 76-2-11. an isotype matched control for LeuSb staining, the bottom panel staining with LO- C02a and a rat isotype matched control for LO-CD2a. FIGURES 29a and 2»b Nucleotide and amino aoid sequences of the L0-CD2a V L chain, a) with leader sequence from MRC vector; b) with leader sequence from LO-CD2a gene. FtOURBS 30a and 30b Nucleotide and amino acid sequences of the L0-CD2a V B chain, a) with leader sequence from MRU vector; b) with leader sequence from LO-CD2a gene. pgmiBd Pøtςriptlpn In accordance with an aspect of the present invention, there is provided a molecule (preferably a monoclonal antibody or fragment thereof) which binds to the same epitope (or a portion thereof) on human lymphocytes aa the monoclonal antibody produced RECTIFIED SHEET (RULE 91) ISA/EP O 94/20619 -10- by the call line deposited as ATCC Deposit No. KB 11423. The antibody which is produced by the dβpoβite cell line is hereinafter sometimes referred to as lC-CD2e. The term 'molecule" or "antibody that binds to the same epitope as LO- CD2a" includes L0-C82a. The tenn , O~CD2a" includes the antibody produced by the deposited cell line A CC HB 11423 and those identical thereto which may be produced, for example, by recombinant technology. The molecules or antibodies of the present invention inhibit human T-cell activation and proliferation and Applicant has found that such inhibition can be effected 'when adding the molecule or antibody either before or after an agent which stimulates T-cell activation. The molecules or antibodies of the present invention have the characteristics of binding to an epitope of a CD3 antigen <CD2 positive human T-cβllβ) but it is to be understood, however / that the ability of such molecules or antibodies to inhibit T-oell activation or proliferation may or may not be effected through binding to CD2 positive calls, although Applicant presently believes that the mechanism of action involves binding of the molecule or antibody to CD2 positive cells. In acoo dance with another aspect of the present invention there is provided a method of preventing and/or inhibiting on-going immune response in human patients through the administration to the patient of an antibody, hereinafter referred to ss O-coaa (or fragment or derivative thereof) or β ny molecule that mi iαβ such antibody or derivative or fragment thereof . A cell line which produces LO-CD2a, was deposited on July 29, 1993, at the American Type Culture Collection, 13301 RECTIFIED SHEET (RULE 91) ISA/EP O 94/20619 -11 - parieiawn Drive, Rockville, KB 20852, and was given the ATCC accession number ATCC H8 114 5. Such antibody is « rat monoclonal antibody. Although Applicants do not want to limit the invention to any theoretical reasoning / it is believed that the mechanisπ. which enables the monoclonal antibody of this invention to prevent or reduce the severity of an immune response, and to inhibit the ectivation and] proliferation of τ-cellβ, is the fact that the O-CD2a antibody decreaβes the density of CD2 expressed on T cell βurfacee and thus decreases the number of CD2* X lymphocytes. It is believed that these mechanisms of action are responsible for not only the prevention of immune response, but also the reduction in severity ot on-going immune responses. In addition, the O-CD2e antibody inhibits natural killer (HK) cell activity in vitro as αxenpli ied herein. This is pertinent to the present invention since it is believed that a non-MHC restricted cytotoxic mechanism such aa KX cell activity has been implicated in graft versus host disease. in accordance with an aspect of the preβent invention there is provided a process for inhibiting initial or further activation and proliferation of T cells in a human patient by administering to the patisnt an effective amount of a molecule (preferably an antibody) vhic binds to the aame epitope (or any part thereof) on human lymphocytes as the 0-CD2a antibody* The preferred molecule is 0-CD2a or a chimeric and/or humanised form thereof. Such a molecule would/ for example, contain the aame complementarity determining region (COR) ae the LO-CD2a antibody. Tht term "inhibit* as used herein throughout this Applicant is intended to mean prevention, or inhibition, or reduction in severity, or induction of tolerance to, or reversal of graft rejection. The tern "graft" aa used herein for purpoaeβ RECTIFIED SHEET (RULE 91) ISA/EP -12- o£ thiβ application shall rαβaπ Any and all transplantation, including but not limited to, allogrβft and xβnogrβft transplantation. sueh transplantation may by way of example include, but not be limited to, transplantation of cells, bone marrow, tissue, solid-organ, bone, etc. The term "immune reιponβe(s)" as used herein is intended to mean immune responses dependent upon T cell activation and proliferation which includes both cellular effects and T cell dependent antibodies which nay be elicited in response to, by way of example and not limitation: (i) grafts, <ii) graft versus host disease, and (iii) autoantigens resulting in autoimmune diseases, which by wey of example include but are not limited to rheumatoid arthritis, systemic lupus, multiple sclerosis, diabetes, mellltuβ, etc. The molecule employed in the present invention is one which binds to the sane epitope (or a part of that epitope) es the 0-CD2a monoclonal antibody. The tern "binds to the sane epitope es LO-CD2a monoclonal antibody" is intended to describe not only the L0-CD2a monoclonal antibody but also describes other antibodies, fragments or derivatives thereof or molecules which bind to the same such epitope aa the 0-CD2β monoclonal antibody. Such other antibodies include by way of example and not limitation rat, murine, porcine, bovine, human, chimeric, humanised antibodies, or fragment* or derivatives thereof. The term "derivative" as used herein means a chimeric or humnissd antibody, single chain antibody, bispecific antibody or other auch antibody which binds to the sane epitope (or a portion thereof) aβ recognised by the LO-CD2a monoclonal antibod • RECTIFIED SHEET (RULE 91) ISA/EP The term "fragment" as used herein means a portion of an antibody, by way of example suoh portior.β of antibodies shall include but not be limited to CDR, Fab, or such other portions, which bind to the earn* epitope or any portion thereof as recognised by O~CD2a. The term "antibody" se used herein includes polyclonal, . monoclonal antibodies as veil as antibody fragments, derivatives as well as antibodies prepared by recαmbinant techniques, such Λβ chimerie or humanised antibodies, single chain or bispecific antibodies which bind to the aame epitope or a portion thereof as recognized by the monoclonal antibody LO-αtfa. The term "molecules" includes by way of example and not limitation, peptides, oligonucleotides or other such compound! derived from any source which minic ths antibody or binds to the same epitope or a portion thereof as the antibody fragment or derivative thereo . Another embodiment of the present invention provides for a method of treating a patient who is to receive or has received a greft transplant with an effective amount of at least one member selected from the group consisting of LO-CD2a antibody, or an antibody, or derivative or fragment thereof or molecules which bind to the sane epitopa (or a portion thereof) as the O~CD2e antibody. The treatment is preferably effected with the whole or intact LO-CD2* antibody. A monoclonal antibody of this invention as hereinabov β described may be produced by techniques known is the art such a β described by ohler and Milβteln (Nature 256, Pc. 495-497, 1973 ) 99 well es the techniques disolosed herein. The preparation of a monoclonal 0-CD2* antibody is described in sore detail in Example 1 of this Application. As herelnabαve indicated I < 0-CD2a antibodies may also be produced by recombinant techniques using procedures known in the art. The rβconbinant antibody nay also be in the form of a chiaeric antibody -wherein the variable or CDR RECTIFIED SHEET (RULE 91) ISA EP regions of a LO-CD2a rat antibody ia combined with the constant ragion of an antibody of another species, Thus, for example, the monoclonal antibody may be humanized by coicbining the variable or CDR region of * rat oCD2 monoclonal antibody vi h the constant region of a human antibody to provide a chimβric human-rat monoclonal antibody. The antibody or molecule of this invention preferably: (i) binds to all T lymphocytes and also to null cells but not B lymphocytes as shown by two color staining of lymphocytes analysed by flow c tome ry (Figures 26 and 27); (11) binds to all T cells (as determined by staining with the aati-CD3 antibody eu4), ell CD4 and CDS positive cells as defined by Leu3a and eu2b antibodies respectively and some lymphocytes which are D3 negative (null cells); (iv) binds to null calls as corroborated by the staining of CD 6 positive cells as detected with Leull, e marker for K cells. (Figure 26); Btaining of B cells, as defined by anti-CDl9 binding, was not seen vith LO-CD2a. (Figure 37). LO-CDJa antibody also preferably has the characteristic that the antibody binds to human null cells, and by double staining has a higher intensity of staining to human cells that are both CD2+ and CD4+ than to human cells that are both CD2+ and CD16+, and haa a higher intensity of staining of human cells that are both CB2+ and CD8+ than to human cells that are both CD2+ end CD 6+. That Lo-CD2a binds to CD2 waa confirmed by transiently expressing CD2 ID COS calls. COS calls were transiently transfeoted with the COM plesnid containing the gene encoding for the entire CD2 molecule, as described in Peterson λ. end Seed B., Nature Volume 329, 10/29/S7, pp B42-846. RECTIFIED SHEET (RULE 91) ISA/EP Tranβfection vas accomplished by the DEkS-dextran method. Cells were harveeted and etβinβd vith the anti-CD2 monoclonal antibody euSb (Bectcn-DicKinson) and LO-CD2a, with murine W632 an antibody to MKC class I as a positive control for staining and vrith the corresponding iiotypβ-matched controls. Specificity of the reeotivity was confirmed by assessing binding of the aa e panel of monoclonal antibodies on CO8 eellβ tranβfβetβd vith an irrβlβvent piaβmid. The staining pattern of theaa monoolonal antibodies on transiently expressed native C03 (Figure 26) indicates that transfee ion with CD2 led to binding of both antibodies, supporting the ability of L0-CD2* to bind to CD2. The preparation of LO-CD2a monoclonal antibody auitatolβ for the purpoaeβ of the present invention should be apparent to those a illed in the art from the teachings herein. An antibody or fragment or derivative thereof or molecule of the type hereinabove described nay be administered j& yXB in accordance with the preeent invention to inhibit the activation and prolifaratioa of T-cells, and deoreaee the deniity of CS2 expression on the cell surface end thereby reduce the number of CD2* T lymphocyte*. Thus, for example, in an in ylvo procedure , euch LO- CD2a antibodies are administered to prevent and/or inhibit immune reepαnee and thereby inhibit T cell activation and proliferation. An antibody or fragment cr derivative thereof or molecule of the type herein above described may be administered *& Umm-tmm ift accordance vith the preeent invention to decrease the denβity of CD2 + expression on the cell surface and thus reduce the number of CD2* cells of the donor cells. By way of example RECTIFIEDSHEET(RULE 1) ISA/EP aπd not limitation, in an ffifc viva procedure, such antibodies or fragments or derivatives thereof or molecules would be infuβed into donor bone marrow prior to transplantation to prevent the onset of graft varsua host disease upon transplantation. In such an 1& vivo or a& l xa. technique, the antibody or fragment or derivative thereof or molecule will be administered in a pharmaceutically acceptable carrier. Ae representative examples of such carriers, there may be mentioned normal ealine solution, buffers, etc, θuch pharmaceutical carriers era veil known in the art and the selection of a suitable carrier is deemed to be within the scope of those skilled in the art from the teachings contained herein. The LO~CD2a antibody or other molecule of the present invention may be administered ia vivo intravenously or by intramuscular administration , etc. As herein above indicated, O-CD2a antibody or other molecule of the present invention is administered in vivo in an amount effective to inhibit graft rejection. The term "an effective amount" for purposes of this Application ahall mean that amount of monoclonal antibody capable of producing the deaired effect, i.e., the inhibition of graft rejection or inhibition of the activation of T-celle. In general, such antibody is administered in an amount of at least 1 mg. It is to be understood that lower amounte could be used. In addition after the initial treatment / the herein above described amounts may be reduced for subaaquant treatments/ If any. Thus the scope of the invention is not limited by such amounts. Zn accordance with the present embodiment, such antibodies are repeatedly administered in order to maintain the inhibition of Φ-eell activation and graft rejection. Thus, by wey of example and not limitation, the antibody may be RECTIFIED SHEET(RULE91) ISA/EP adminietared by intravenous infusion over a one to two hour period in amount of from about 1 mg/dose to about 50 mg/dose in a. physiologically acceptable carrier suspension once or twice a day for a period cf from about eight days or more, as needed. Such treatment for graft rejection iβ preferably started at / or immediately prior to, or shortly after transplantation or when graft rejection occurs. The treatment could be given once or tvice a day for Λβ little as one or tvo days whan started at the time of transplantation to induce a selective hyporeβponsive atate to the transplant. Such treatment for autoimmune diseases with respect to the administration of the antibody or fragment or derivative thereof or molecule in accordance with the present invention is begun when the attending physician has determined it is desirable to inhibit a pathological immune response. The techniques of the present invention for inhibiting the aetivation of T-cellβ nay be employed alone or in combination with other techniques, drugs or compounds for inhibiting the activation of τ~cell or inhibiting graft rejection or graft versus host disease. The invention will be further described with respect to the following examples, which are illustrative and which are not intended to limit the scope of the invention. The cells, cultures, nλba and mitogenβ uβed in the examples may be prepared and uβed by processes and procedures known and practiced in by those of ordinary skill in the art. The following is an example of a preoess or procedure that may be used for the preparation and use of the oells r culturee, ΛAbi and aitogene used in the examples which follow. Calls aad crttlt rti PBMC were obtained by Fieoll-Hypaque (Pharmacia, Sweden) sedimentation of heparinised blood obtained from the RECTIFIED SHEET (RULE 91) ISA/EP local aiood Donor Center. Isolated PBMC were reβuspended in enrlched inadium RPMI 16_0 medium (Oibeo, Belgium), supplemented with 100 0/ιr.l penicillin, 100 fig/ml βtrβp omycin, 20 mM L- Glutaminβ, and 20% pooled human AB serum or 15% heat'inactivated fetal calf serurc. PBMC were cultured at IxlC 1 ce la/well in 96 U-well micro plates (Falcon) in a final volume o£ 20C μl of culture medium/well. Bidirectional KLC were performed with 1x10 s cells of each donor/well in the same volume o£ culture medium aa noted above. All culture! were made in triplicate. Bight hours before the timea indicated in the reaulte, cultures vere pulsa- labelled with 2.0 μci/wβll of 3„-T (Amershan, Belgium; 247.9 GBq/mnol; 6.7 Cl/rπmol) end the radioieotope incorporated in culture* vaa quantified by liquid scintillation in a Betacounter (Bβckman L3 6000 SE) . The percentage of inhibition wad calculated aa follows* % Inhibition •» [1- (mean cpm of tested culture/mean σpm control culture) ] xioc. All reeulta are eκpreaeed at the mean of three independent cultures. Standard deviation was alvaya leaβ than 15% of the mean, except for thoae caaes where theee values are indicated on the graphics. Cytofluoroaβtric analyses vere performed using a FACScan cytofluoro graf (Becton Dickinson) with Hewlett-Packard hardware equipped with the Consort 30 program, independent analyaia of staining of lymphocytes and blast-calls was possible using differential gating as defined by βise and granularity. 25 000 events were analyzed for each sample« In theee experimenta, O-CD2a final concentration was 300 ng/ml, except when indicated. Mabe and Mi gyπ LO-DRA and L -τact-1 (both riTC-labelled) , are rat mλbs produced in our laboratory (op. eit. H. Basin <Bd) I960 p.2B7). LO-ϊact-1 is directed against the p55 chain of the IL-2 receptor (op. oit. K. Basin Immunol. 1984 and Jansten, M. , Buck, o. and RECTIFIEDSHEET(RU £91) ISAEP Malno r V.C. in Leucocyte Typing IV White Cell Dif arβntiation Antig-ma, w. Knapp (ED), Oxford Enivβraity Presa, 1989, p.403). Mouie anti-human-CD2 and anti-CD3 mλba ( eu-5b and eu-4a-Firc- labelled) were obtained from Becton Dic-tinaon (Belgium). Moutβ anti~human-CD4 or anti-human-CD8 mAbβ (phcoβrythrine-labelled) , and mouse Igβ FITC- or phceβrythrinβ-labelled (negative controls) were obtained from Coulter. OKI3 (Ortho-Cilaς, Belgium) was uaed et a final concentration of 100 ng/ml- Phytoheraβgglutinin k (PHλ; Wellcome Labi, UK) and Coaeanavelln A (Con A; Calbiochβm Co., USA) were used at a final concentration of 1 and 10 μg/ml, rββpβctivβly. BJPti iYltt ifln of L -CD2t> The concentration of purified LQ-CD2a was adjuβted to 1 mg/ml in 0.1M eodi bicarbonate buffer, pH β.4.NHS-biotift (Boahringβr Mannheim 1008 960) wee diieclveύ in 2X00 at a concentration of 1.5 mg ml. For each KAB, 0.1 ml of NHS-biotin aolution waβ added. The mixture wet rotated for 2 hri. at ambient temperature. The reaction ae completed by adding 0.1 ml of 2M trie-KCL, pB 8.0, for each ml of antibody (10 minutes at ambient temperature), followed by 1 ml of 1% BβA in phoaphate buffered •A ine (PB3) for each ml of antibody. To remove free bio in, the eolution was dialyaed overnight at 4"C in 1O00 volumes PBS. Both the biotinylation reaction and the conjugated mλb ware shielded from light by covering with aluminum foil. Ly!* of red blaorf cell, tmc . *BC were removed from whole blood by lyβia with ammonium chloride. A 10X atoci solution was prepared which conβieted of 90g NH 4 Cl lOg KKCO,, 370 mg BDTA, and H j O to a volume -of 100 mil. Forty lβ of IX ammonium chloride was added to each 10 li of blood and incubated for 10 min. at room temperature. The mixture wa§ then centrifugad at 1200 rpiα for 10 min and the pellet reeuapended in 10 ml PBS with 0.11 azide. RECTIFIED SHEET (RULE 91) ISA/EP Btaininψ of peripheral blood, staining was carried out in round- bottom 96 well clutter plateβ (Coβtar #3790) at 4*C. Tor single color ataining, ten μl of λb was appropriately diluted in PBS containing 0.2 mg human immunoglobulin and added to each well. Red blood cell depleted blood was distributed into plate* at a volume of 90 μl per vail. Cell* and mAb were mixed by gentle tapping and incubated 30 min. Fifty μl of cold PBS vaβ added to each well and piat i were centrifuged at 1900 rpma for 2 min. Supernatant vaβ discarded by inveriion and gentle flicking of the plate. Cells ware diape sad by tapping the plate on the counter. The wash procedure wee repeated twice by adding 200 μl of cold PBS. Ten μl of a 1/20 dilution of goat F<αb') a anti-rat lg-FITC waa added to the dieperβed cells in each wall and incubated for 30 min. in the dark. Cells were washed by the addition of 180 μl of cold PBS to each well followed by centri ugation at 1900 rpmβ for 2 min. supernatant was discarded, cellβ dispersed, and 200 μl of cold 0.5% pβraformaldfch.yde waa added to each well. Cells were trana erred to tubes (Talcon #3054) and diluted to approximately 0.5 lβ with 0.5% paraformaldβhyda. Samples were evaluated en a Bβcton-Dickinaon FACβcan machine using LTSI β II ee ware. Dual color staining waa carried out by a similar protocol. After cells ware incubated with the primary Ab and the FITC-co jugated anti-rat reagent , a 1/5 dilution of normal ouaa serum was added to block any remaining aicea on the anti- rat reagent. Following α 15 min. incubation (no wash), 20 μl of a Pi-labeled mAb specific for a known CO determinant was added and incubated for 30 min. Cells were washed and fixed as described for single staining. RECTIFIED SHEET (RULE 91) ISA EP EXAMPLE 1 LO-CD2a iε a rat (IgG2b-Kappa) anti-CD2 monoclonal antibody produced and characterized in our laboratory as indicated elsewhere (See the following references: Xia, H., Ravoet, A.M., Latinne, D., Ninanne, J., De Bruyere, M., So al, G. and Bazin, H., in H. Bazin (Ed), Rat Hybridomaε and Rat monoclonal Antibodies, CRC Press, Inc., Boca Raton, Florida 1990, p.309 and Ravoet, A.M., Latinne, D., Seghers, J., Manouvriez, P., Ninanne, J., DeBruyere, M. , Bazin, H. and Sokal, G.- in H. Bazin (Ed) Rat Hybrido as and Rat Monoclonal Antibodies, CRC Press Inc., Boca Raton, Florida, 1990, p. 287). LO-CD2a was purified from ascitic fluid by immunoaffinity chromatography taking advantage of the allotypic difference existing between the immunoglobulins of the rat receiving the producing hybrido a and the mAb secreted by the latter (Bazin, H., Cormont F. and DeClercq, L.. J. Immunol. Method. 1984, 71:9). It recognizes the total population stained by the mouse mAb Leu-5b (FITC-labelled) Figure 1 and roughly 90% of the population marked by the mouse Til (Rhodamine-labeled) mAb ((data not shown) (see FIG. 1)). The epitope recognized by LO-CD2a on the CD2 molecule, is different from the epitopes recognized by the anti-CD2 mouse mAbs Leu-5b and Til (Figure 2). EXAMPLE 2 LO-CD2a exhibits modulatory but not mitogenic effects on PBMC In order to determine the effects of the rat mAb LO- CD2a on resting lymphocytes, PBMC were incubated in the presence of increasing concentrations of this mAb. As can be seen in Table 1, PBMC incubated during 6 days in the presence of LO-CD2a show no significant variations in the rate of 3 H-T incorporation as compared with control cultures. Cell viability at the end of this period was variable but averaged around 80% as assessed by trypan blue exclusion. When resting PBMC were incubated in the presence of LO-CD2a, there was no significant variation in the phenotypic expression of several membrane markers, as assessed by flow cyto etry. Cellular markers of resting mature T-cells (such as CD3, CD4 and CD8) show the same pattern of variation during 6 days of culture in the presence or in the absence of LO-CD2a, and activation molecules such as CD25 (IL-2R/p55) are not expressed in these experimental conditions or are not modified by LO-CD2a as is the case of DR antigenic determinants. (Figure 3) When PBMC were incubated for 6 days in the presence of LO-CD2a, a significant decrease was observed in the percentage of Leu-5b+ gated lymphocytes. (Figure 4) The percentage of CD4- and CD8- lymphocytes is not affected during a 6-day period of cultures by the presence of LO-CD2a, indicating that the observed decrease of CD2-bearing lymphocytes cannot be attributed to an elimination of these cells but rather to a disappearance of the CD2 molecule or to a conformational change in this glycoprotein produced by the binding of LO-CD2a. In order to verify if the observed decrease in Leu-5b- ly phocytes was due to a conformational change of CD2 or to a disappearance (internalization or release) of this molecule after the binding of LO-CD2a, PBMC were cultured in the presence of 500 ng/ml of LO-CD2a and analyzed through 6 days in flow cytometry using Leu-5b (FITC-labelled) , Tll-RDl (Rhodamine-labelled) and MARK-3 (FITC-labelled) . As shown in Figure 5a, Leu-5b or Til mabs are not able to bind to PBMC after 2 to 4 days of culture in the presence of LO-CD2a. Under these conditions, the mouse anti- rat kappa chain mAb MARK-3 labelled 50% of cells at day 6 of culture indicating that only 35% of the original CD2-bearing cells show no LO-CD2a on their surfaces, yet Leu5b-FITC and Tll- RDl staining have decreased markedly at day 2. This suggests that a conformational alteration of CD2 rendering the epitope of Leu5b and Til unavailable for binding occurs in response to LO- CD2a. The analysis of the mean fluorescence of CD2+ cells indicated that the density of expression of this marker decreased with time in the presence of LO-CD2a. The same phenomenon was observed whether Leu-5b FITC-labelled or LO-CD2a (revealed by MARK-1 FITC-labelled) were used to detect the CD2+ ly phocytes. Aliquots of the same PBMC were cultured in parallel in the presence of Leu-5b (commercially available mAb, dialyzed against PBS, 1:2 final dilution in culture medium). As shown in Figure 6b, in those experimental conditions all the CD2-bearing cells are coated by the Leu-5b mAb (as revealed by goat anti- mouse-FITC). Staining by Tll-RDl was markedly reduced, whereas a smaller, slower decrease was observed in the percentage of cells presenting the epitope recognized the LO-CD2a-FITC mAb. Taken together these results indicate that CD2 molecules have partially changed their conformation in response to LO-CD2a, and that a slow modulation of CD2/LO-CD2a occurs. As determined by flow cytometry, the number of blast-cells present in unεtimulated PBMC at day 0 of culture, remained constant (200 to 300 blast-cells over 25,000 events) or decreased during the period of study (both in the presence or in the absence of the rat mAb), indicating that no blastogenesis waε induced by the presence of LO-CD2a. LO-CD2a inhibits MLR When MLC were performed (over a period of 6 days) in the presence of increasing concentrations of rat mAb, a significant inhibition of the MLR (as measured by 3 H-Thymidine ( 3 H-T)-incorporation) , was observed at concentrations of mAb as low as 125 ng/ml. In Figure 6a, we show a typical example of a dose-response curve of MLR inhibition by LO-CD2a. As can be seen in this Figure 6a, LO-CD2a induces 80% inhibition of MLR (6 days of culture) at 250 ng/ml and this percentage of inhibition remains almost constant or higher than 80% over a wide range of concentrations (0.25 to 5.0 μg/ml of mAb). Figure 6b shows a time-course of the inhibitory effects of different concentrations of LO-CD2a on MLR from day 0 to day 6 of culture. A typical example of 3 H-T-incorporation on MLC (in the presence or in the absence of LO-CD2a) is shown in Figure 6c, where LO-CD2a was added at a final concentration of 200 ng/ml. In Figure 6d we show the effects of LO-CD2a on MLR, when this mAb (at 200 ng/ml) is added at varying times after initiation of MLC. More than 90% inhibition of MLR (as measured by 3 H-T incorporation) is obtained when this mAb is added at day 0, and this inhibitory effect is still present (45% inhibition in this example) when L0-CD2a is added 4 days after the beginning of MLC. Similar results (not shown) were obtained with higher concentrations (from 0.20 to 5.0 μg/ml) of LO-CD2a. LO-CD2a blocks the pathway of IL-2R expression When cytofluorograph analyses were performed on the lymphoblaεt subset of a MLC (Figure 7a and b), the following observations were made: a) the number of blast cells (around 300- 500 blast cells of 25,000 events analyzed) already present at initiation of MLC rose sharply from day 4 to day 6 in control cultures (more than 1200 blast cells from 25,000 events analyzed); b) in MLC performed in the presence of LO-CD2a, there was no significant variation in the number of blast cells during the whole period of culture and at day 6 the number of blast cells is always lower or nearly the same as the initial number of blasts at day 0 (Figure 7a); c) the percentage of CD25 blasts rose sharply among cells incubated without LO-CD2a (Figure 7b); d) this percentage remains below 20% in the small number of blasts from the MLC incubated in the presence of mAb (Figure 7b) , and the mean fluorescence (as a measure of CD25 expression) decreased by 75% as compared with blasts present in control cultures (results not shown); e) in the absence of mAb the percentage of CD3- blasts remains constant during the first 4 dayε of culture (Figure 7b) and on day 6 the percentage of CD3- cells increased to 90%, while in the presence of LO-CD2a the percentage of CD3- rises slowly to reach only about 45% at day 6. Theεe reεultε indicate that the preεence of LO-CD2a inhibitε the entrance of these cellε in the pathway of activation characterized by the expreεsion of IL-2 receptor (CD25). The number of CD2+ blasts remains constant or decreaseε in the presence of LO-CD2a, and the density of expression of this membrane marker is strongly diminished under these conditions (data not shown). When phenotypic analyses were performed through 6 days on the resting (non blast) lymphocyte subset of MLC, results similar to those described in Figure 3 were obtained: in the presence of LO-CD2a, no significant variation could be detected in the percentage of CD3+, CD4+ or CD8+ lymphocytes, as compared with control cultures (Figure 8); no CD25 expression (activation marker) could be detected whether in the presence or in the absence of LO-CD2a during 6 days of culture. These results suggest that LO-CD2a has a very weak, if any, effect on the resting subset of T-lymphocytes in MLC; that is to say, in T- cells not committed in the process of activation. At the same time, as shown in Figure 4b, LO-CD2a induces a significant decrease in the percentage of CD2+ lymphocytes during MLC. This phenomenon is accompanied by a drastic decrease of the mean fluorescence as detected by the Leu-5b mAb. LO-CD2a can block the pathways of T-cell activation dependent on the TcR/CD3 complex or on mitogen receptors. When LO-CD2a was added to mitogen-activated PBMC, a significant inhibition of 3 H-T incorporation was observed. In one of three experiments of PBMC incubated with mitogens (OKT3, ConA and PHA) in the presence or in the absence of LO-CD2a added either at time 0 or 1 hour af er the start of cultures. In the first case, mitogens were added 1 hour later. When LO-CD2a was added 1 hour after the initiation of culture, mitogens were added at time 0. This was done in order to know whether, preincubation of PBMC with mitogens or LO-CD2a could trigger events that could be affected by the addition of the second reagent. Cultures were harvested at 96 h, after a pulse-labelling (6 h) with 3 H-T. More than 50% inhibition of 3 H-T. incorporation was observed in the presence of LO-CD2a, whether it is added firεt or after mitogens. (Figure 9) The same effect was obεerved when cells were harvested 4 days after the onset of MLC and exposed to mitogens (results not shown). A drastic decrease in 3 H-T incorporation was observed two days after the onset of MLC, in those cultureε receiving both the mitogen and L0-CD2a, aε compared with the εame cultures receiving only mitogen (results not shown). Preincubation of MLC with LO-CD2a before addition of mitogen, lowered the 3 H-T-uptake to values comparable with MLC without OKT3. LO-CD2a was also able to inhibit mitogen induced proliferation if added one day after the initiation of mitogen induced proliferation. The results of experiments performed with two donors are shown in Figure 10. PMBC were incubated along with mitogens (OKT3, ConA and PHA). In these experiments, LO- CD2a was added either at time 0 (Day 0), 24h (Day 1) or 48h (Day 2) after the start of the cultures. The inhibition of proliferation in response to OKT3 and ConA by LO-CD2a was significant if added 24 hours after the addition of mitogen at time 0. EXAMPLE 3 Inhibition of Natural Killer Cell (NK) Activity. PBMC were isolated from heparinized blood by Ficoll Hypaque Sedimentation. After washing, the effector cells. suspended in enriched medium, were incubated overnight at a concentration of lX10 6 /ml in a Falcon plate to eliminate the monocytes (by adherence). The target cells (K562 cell line) were labeled by overnight incubation with chromium Cr (0.9 ml of a cell suspension at 3xl0 6 /ml + 0.02 ml from a solution of 5mCi/ml Cr, Amersham) . After a 16-hour incubation, effector and target cells were washed four times, counted and incubated in a 96 V bottom microplate at different E/T ratios: 200/1 (100 ul of a suspension of 4xl0 6 /ml effector cells with 100 ul of 2xl0 4 /ml target cells) 100/1, 50/1 and 25/1. After a four-hour incubation, the 51 Cr release was measured by counting 100 μl supernatant from each well in a gamma counter. Maximum (target cells + HCLIN) and spontaneous release (target cells + enriched medium) were used to calculate the specific lysis: test- spontaneous release %Specific lysis = X 100% maximum - spontaneous release Inclusion of LO-CD2a at 5, 1 and 0.5 ug/ml in the NK assay with two normal donors (Figures 10a and 10b) led to an inhibition of cytotoxicity of approximately 50% with all tested concentrations of antibody and over all tested E/T ratios. This is in comparison with essentially complete inhibition of proliferation in the MLR at doses at or above 0.25 ug/ml. EXAMPLE 4. IN VIVO STUDIES IN NON-HUMAN PRIMATES MATERIAL AND METHODS Monoclonal Antibodies MARK3-FITC is a mouse mAb directed against the rat lg kappa lb allotype conjugated with FITC. MARG2b-biotin is a mouse anti-rat lgG2b immunoglobulin mAb conjugated with biotin. These two mAbs were produced and labeled in our laboratory. For i munofluorescent tests they were used at a final concentration of 2.5 μg/ml. Leu-5b-FITC (Becton-Dickinεon) and Til Rhodamine (COULTER) are two mouse anti-human CD2 mAbs. T4- and T8- Rhoda ine-labeled (COULTER) are mouse anti-human CD4 and CD8 mAbs respectively. Phenotype Analysis Anti-human T-cell mAbs (anti-CD2, -CD4, -CD8, see above) were added to 100 μl samples of whole blood and incubated at 4°C for 45 min. Red blood cells were lyεed with a Triε-buffered ammonium chloride-lysing buffer (144 mM NH 4 CL j /17 mM Tris, pH 7.2) and lymphocytes were washed with PBS/2% FCS/0.2% NaN3. For detection of non-labeled mAbs, a second mAb (FITC- or biotin- conjugate) was added to a final concentration of 2.5 μg/ml. After 45 min. incubation at 4°C, cells were washed with PBS/FCS/NaN3. For biotinylated mAbs, a further incubation (15 min) with Streptavidin-Phycoerythrin conjugate was done. Labeled human or monkey lymphocytes were reεuεpended in a 2% formalin solution and analyzed in a FACSan cytofluorometer (Becton- Dickinson) equipped with the lysis II program for gating lymphocytes as a function of εize-vs-granularity. As a control for nonspecific staining, aliquots of cells were incubated with FITC- or Phycoerythrin-conjugated mouse lgs (Coulter). Level of circulating Abs LO-CD2a in serum was quantified by ELISA using a mouse anti-rat lgG2b mAb (MARG2b-8, produced in our laboratory ) as firεt layer (coating) and a mouse anti-rat kappa chain (MARK-3) mAb coupled to horseradish peroxidase for detection. Briefly, microtiter plates (Falcon) were incubated overnight with 100 μL/well of MARG2b-8 (5μg/ml) and unoccupied sites on plastic were saturated with PBS containing 5% powdered milk (bovine). After 1 h incubation at room temperature, plates were washed with PBS with 0.1% Tween-20, and incubated 1 h with lOOμl/well of diluted monkey or human serum. After washing out unbound material, plates were incubated 1 h with 100 μl/well MARK3-peroxidase (2 μg/ml in PBS). After washing again, plates were incubated with OPD (o-phenylenediamine dihydrochloride, 0.4 mg/ml, Sigma Chemicals), in citrate-phosphate buffer containing 0.03% H 2 0 2 . The colored reaction product was detected at 492 nm. A standard curve was made in parallel with a known concentration of purified LO-CD2a serially diluted in a pool of control monkey or human serum. The detection of monkey or human anti LO-CD2a antibodies was performed by ELISA using 96 well micro plates coated with LO-CD2a (5 μg/ml). Anti-LO-CD2a human or monkey antibodies bound on the plates, were revealed by horse-radish peroxidase labeled rat anti-human IgM (LO-HM-7) or IgG (LO-HG-22) mAbs. A. CYNOMOLGUS MONKEYS One Cynomolguε monkey received 10 mg/day of LO-CD2a for three consecutive days. The monoclonal antibody was well tolerated. A lymphocyte depletion was observed after the first injection but a very low additional depletion was obtained after the 2d and 3d injections. The second monkey received 20 mg/d for 10 days. The mAb was also well tolerated. No side effects were observed after dosing in that the animals were active, alert, eating well with no evidence of nausea or gastrointeεtinal disturbance. The lymphocyte counts and cell populations in the second monkey are summarized in Figures 19 and 20. The NK activity was slightly reduced after the 10 injections (Figure 21). The circulating levels of MAb were very high (Figure 22) and immunization occurred at the end of the treatment (Figure 23). B. BABOON The experiment described here was undertaken to determine the tolerance of a baboon to LO-CD2a, to analyze the effects of this mAb on some of the membrane markers of baboon lymphocytes and to determine the half-life of LO-CD2a in εerum. Staining of baboon cells with LO-CD2a reεults in <20% positive cells at a mean fluorescence intensity significantly lower than that of stained human cells, consistent with modest cross reactivity with baboon cells. The εtudy was done on a male baboon (papio mormon) weighing 8.8Kg. Before each injection of LO-CD2a the monkey waε anesthetized; the firεt time with Ketaler (2 ml) and Prazine (0.5 ml), the second time with Ketaler only and the subsequent times with ketaler and Prazine (0.3ml). LO-CD2a was injected intravenously (i.v. in 10 min.), diluted in 100 ml of physiological serum. For phenotypic analysis of lymphocytes and measurement of circulating antibodies (injected L0-CD2a and newly formed anti-L0-CD2a antibodies, and preexisting cross-reacting baboon anti-L0-CD2a antibodies), blood sampleε (10 ml) were taken in two tubes. The tube for lymphocyte typing contained EDTA. Samples were taken prior to the first treatment to determine baεeline levels. The firεt does (10 mg) of LO-CD2a was administered on day 0 of the study; the four following doses (10 mg/dose) were administered on days 7, 8, 9, and 10. Blood samples were taken a few minutes after each LO-CD2a dose. On dayε 7 and 9 a supplementary blood sample (in an EDTA-containing tube) was also taken before the LO-CD2a injections. Blood samples were taken on days 1, 2, 11, 12, 13, 16 and 24. No abnormal reactions in activity or feeding habits were observed during LO-CD2a injections or throughout the period of εtudy. The weight of the animal remained around the 8.8 Kg meaεured at day 0 (see table below). Weight of baboon (in Kg) from day 0 to day 24 Day 0 7 8 9 10 12 13 16 24 Weight 8.8 8.9 9.1 9.1 8.8 9.0 9.1 8.9 8.9 ANALYSIS OF PHENOTYPE AND CIRCULATING mAb The fluorescent staining of this baboon's peripheral blood lymphocytes revealed some interesting features: a) Under the effect of LO-CD2a the CD2-positive subset of lymphocytes decreased significantly (as revealed by two different anti-CD2 mAbs) at the end of the 5th dose of LO-CD2a, that is at the time of a maximal accumulation of mAb in blood (see Figures 17a and 17b). Given that the CD4+ and the CD8 + subsets of lymphocytes do not decrease during this period (Figure 19), and because the CD4+ and CD8+ cells comprise most of CD2 bearing lymphocytes, the decrease in CD2 + cells indicates that it is the membrane marker expression that is decreasing, rather than the lymphocytes. As can be seen in (Figure 17a), a slight decrease of CD2+ (Leu-5b + or Tll + ) positive lymphocytes is observed after the first dose of LO-CD2a. Two days after this first dose, the level of CD2 positive cells (Leu 5b + of Tll + ) rose to the starting values. At the end of the four 24-hour spaced doses of LO-CD2a (days 7-10) the percentage of CD2 poεitive cellε (Leu 5b + or Tll + ) decreased sharply and began to rise slowly 3 days after the end of the LO-CD2a administration. b) At the same time, the percentage of LO-CD2a poεitive cells, that is, the percentage of cells bound by the circulating mAb rose to 22% after the 2nd dose of LO-CD2a (day 7) and then decreased as did the CD2 + cellε revealed by the anti-CD2 mAbs Leu-5b and Til (Figure 17). The decrease of LO-CD2a+ cells was revealed by the MARK-3FITC mAb (Figure 18). The decrease of LO-CD2a+ cellε was determined by detection of the LO-CD2a present on cellε as detected by MARK3- FITC or by the MARG2b-8-biotin conjugated mAbs (Figure 18a). The same phenomenon was observed if cells were first incubated with L0-CD2a at 2.5 μg/ml to saturate all the sites unoccupied by circulating mAb. LO-CD2a was detected by MARK-3-FITC or MARG2b- 8-biotin (Figure 18). c) As can be seen in (Figure 19), the T4 positive subset of baboon lymphocytes showed a moderate rise during days 9 to 12 after when the percentage of T4 + lymphocytes returned to its initial value. Conco itantly with the rise in T4 + cellε, the percentage of T8 positive lymphocytes rose from day 9 to day 11. After that day this percentage returned to initial values . d) The levels of circulating mAb LO-CD2a decreased to background values 3 days after the firεt injection (εee Figure 17b) . When LO-CD2a is applied in four short-time spaced doses (days 7 to 10), the levels of serum LO-CD2a (around 3.7 mg/ml, maximal value in this period) decreased εlowly after the last dose (days 10 to 16), indicating a relatively long half-life of the Ab in this animal model. No baboon anti-LO-CD2a antibodies were detected in the blood samples collected on days 11, 12, 13, 16 and 24. CONCLUSION L0-CD2a seems to be well tolerated by non-human primates, aε demonstrated by the absence of apparent reactions in cynomolgus monkey baboon. L0-CD2a εeemε to have a relatively long half-life in the baboon. Twenty-four hourε after the firεt doεe of LO-CD2a (day 1 in Figure 24b), 50% of the maximal detectable level of MAb waε still present in serum. Three days after the last dose of LO-CD2a (day 13 in Figure 24b), 50% of the maximal detectable level of mAb was still present in serum. On the other hand, the decrease in the percentage of CD2 poεitive lymphocyteε followed by a εlow riεe of this percentage of cellε, show the same kinetics observed in human PBMC mononuclear cells cultured in the presence of LO-CD2a. Example 5 Patients treated with LO-CD2a Patients treated with LO-CD2a on a compassionate basis PATIENT #1 (Mb . E . ) This was a female patient with chronic pyelonephritis, who was treated with a renal allograft for end-stage renal failure. A rejection crisis occurred and was treated with 10 days of 0KT3. The creatinine level dropped from 2 to 1.4 mg/dl. Approximately four (4) months later a rejection crisis was diagnosed by a creatinine level of 2 mg/dl and a biopsy indicating moderate rejection. The patient was treated with 1.5 g Solu edrol and a course of ATG for the following eight (8) days at which time the creatinine level was 1.65 mg/dl. Seven days after treatment a biopsy was performed and indicated cellular rejection and moderate vascular rejection. Two days after the biopsy (day 0) the patient was anuric with a creatinine level of 2.4 mg/dl. That εame day the patient received 10 mg of L0-CD2a, 1.5 g Solumedrol, plus 1 g Polarimin (an anti-histamine) and 1 g Dafalgan (acetaminophen). No side effects were noted. By the end of 23 hours, the patient produced 700 ml of urine and the creatine was 2.72 mg/dl. For the next 9 days she received 10 mg/day of L0-CD2a. The patient left the hospital without a follow-up biopsy at that time, Day 11. Measurement of serum creatinine level during ATG treatment and during the following LO-CD2a treatment indicated the creatinine level rose despite ATG treatment and fell and stabilized with LO-CD2a (Figure 27). The leukocyte count fell from a high of 10,000 to 2,000 during the treatment with LO-CD2a and continued to fall until the last measurement on day 21 (Figure 20). The lymphocyte count was low and variable during the period of observation. The serum levels of L0-CD2a rose to peaks of 2.0-3.0 μg/ml immediately following each treatment and fell to lows of approximately 1.0 μg/ml between each treatment (Figure 21). With the last treatment on day 9, the level fell by 50% in 24 hours and to 0 by day 14. This patient returned to the clinic on day 40. The patient's creatinine level was 2.27 on day 40, 2.48 on day 50 and rose to 3.11 by day 66 at which point a biopsy was obtained, with the initial report consistent with severe cellular rejection and interstitial hemorrhage (see below). The patient was treated with 150R of irradiation to the kidney, 3 x 125 mg Solumedrol, while continuing on maintenance therapy of cyclosporin plus 12.5 g/day of steroids. The creatinine level continued to rise during the subεequent period. On day 70 the creatinine level was 3.3; day 80, 5.63; day 84, 8.35. By day 86 the creatinine level was 10.8 and a transplant nephrectomy was performed on day 88. This patient'ε compliance-with maintenance immunosuppreεεion during the period between her discharge on day 10 and her biopsy on day 66 is in question and the loss of the kidney despite the evidently successful rescue must factor in the uncertain compliance. (ii) PATIENT #2 The patient waε a 38 year old male who was Hepatitiε C + . He had received a renal allograft for the treatment of end stage renal failure due to chronic interεtitial nephropathy. One year and three months later, he underwent a transplant nephrectomy due to acute cellular and vascular rejection resistant to a course of OKT3. One year and ten months from the transplant nephrectomy, he received a second renal allograft. Three days later hiε creatinine level waε 1.4 mg/dl. Three dayε later he received 500mg Solumedrol; the patient's creatinine later that day was 1.8 mg/dl. On the following day he received 500 mg of Solumedrol; creatinine was 3.25 mg/dl. The following day he received 500 mg Solumedrol; his creatinine waε 2.95 mg/dl. Three days later his creatine level was 2.3 mg/dl and he underwent a biopsy which demonstrated 3 plus cellular rejection. Three days later he received 10 mg LO-CD2a, plus 200 mg Solumedrol. Polaramine and Dalfagan. Side effects observed were limited to εleepineεε; no hyperthermia or hypertension were noted. For the next 9 days he received daily treatments of 10 mg of LO-CD2a. The day following the end of such treatments a biopsy showed no signs of rejection. The patient tolerated the courεe of LO-CD2a well with no evidence of clinical εide effectε, including no fever or hypertension with any dose. Routine hematological and clinical chemistry laboratory tests (including LFTs) obtained during the courεe of treatment demonεtrated no alterationε attributable to the adminiεtration of the antibody, except for a decreaεe in the lymphocyte count from 290/cubic mm to a low of 100/cubic mm and the reduction in creatinine level associated with resolution of the rejection crisis (from 2.7 mg/dl at the initiation of treatment with LO-CD2a to 1.10 at the end of the course). Figure 22 shows the serum creatinine level of this patient, aε falling from 2.5 to approximately 1.0 on the days following treatment with LO-CD2a. The patient was lymphopenic prior to and during treatment and the leukocyte count showed no dramatic alteration with treatment (Figure 23). In this patient the serum levels of LO-CD2a did not rise above 2.0 μg/ml after each treatment and fell to lows of 1.0 to leεε than 0.25 μg/ml (Figure 24). Eight monthε after the firεt treatment with LO-CD2a the patient was doing well with normal renal function and no evidence of recurrent rejection. Patient 2 - Biopsy #1 - Diagnosis: Indeterminate. The biopsy contained about 20 glomeruli which are unremarkable. There was a sparse mononuclear infiltrate with a minor degree of interstitial edema. Only minor degrees of tubular invasion were found and no vascular lesions. These findings are insufficient for the diagnosis of acute cellular rejection. There were rare mononuclear cellε in small arteries, which were suspicious, but did not meet the criteria for the diagnosis of rejection. Patient 2 - Biopsy #2 Approximately 2 weeks after the first biopsy - No diagnostic abnormality recognized. The biopsy looked similar to the previous biopsy and contained about 10 glomeruli. The infiltrate waε very sparse and no vascular lesions were identified. (iii) PATIENT #3 The patient waε a 19 year old with von Willebrand disease who received a renal allograft for the treatment of end stage renal failure due to chronic pyelonephritis. The transplant waε removed on 17 days later due to acute vascular rejection with secondary hypertension after failure of a 6 day courεe of OKT3. Five and one-half months later he received a second renal allograft. Ten days later his creatinine level was 6 mg/dl. The next day the creatinine level waε 7 mg/dl and a biopεy indicated 3 plus cellular rejection and vascular rejection (proliferative endarteritiε without necrosis or thrombosis). That same day he received 10 mg of LO-CD2a, 40 mg Solumedrol and Polaramine and Dafalgan. No side effects were observed. For the next 9 days he received daily treatmentε of 10 mg of LO-CD2a, with no other drugs and no side effects. Two days after completion of the treatment, his creatinine level was 1.75 mg/dl and a biopsy indicated no sign of acute rejection, with interstitial necrosis and one focal spot of chronic rejection. No clinical side effects (alteration in BP or temperature) were observed. Routine hematological and clinical chemiεtry laboratory teεts (including LFTs) εhowed no changes attributable to administration of the antibody except the decrease in creatinine level associated with the resolution of the rejection crisis (from 7.10 mg/dl on the initiation of treatment with L0-CD2a to 1.75 mg/dl at the end of the 10 day course) . The lymphocyte count was 340/cubic mm prior to treatment and fell to a low of 220/cubic mm during treatment, rose to 690/cubic mm 9 days after cessation of treatment with the LO-CD2a and had risen to 1000/cubic mm 23 days after the end of treatment. The leukocyte count in this patient waε not significantly altered by treatment (Figure 25). The serum creatinine level fell dramatically with treatment (Figure 25). Seven months after the first treatment with LO-CD2a the patient was doing well with normal renal function and no evidence of recurrent rejection. Patient 3 - Biopsy #1 - Diagnosis: Severe cellular rejection affecting small arteries and to a lesser degree the interstitium and glomeruli. An arcuate sized artery showed a marked mononuclear infiltration of the intima with disruption of the elaεtica. There was sparse infiltrate in the interstitium, which occaεionally invaded tubules. The interstitium showed diffuse, mild interstitial edema. There were about 7 glomeruli present. These show hypercellularity with mononuclear cellε and endothelial swelling. Overall, thiε pattern was diagnostic of severe, accute cellular rejection. Patient 3 - Biopsy #2 Approximately 2 weeks after the first biopsy - Diagnosis: Consistent with treated rejection. The biopsy showed a few small arteries, which show intimal fibrosis sometimes with a mucoid material but a very minimal cellular infiltrate. The interstitium showed a fine diffuse fibroεiε and a minimal mononuclear infiltrate. Tubules were locally atrophic but otherwise unremarkable. There was no evidence of active cellular rejection. (iv) PATIENT #4 The patient who was suffering from severe graft versus host disease (severe skin, gut, renal and CNS toxicity resistant to high dose predniεone) after an allogeneic bone marrow transplant received 12 days of LO-CD2a at 10 mg/day. His symptoms improved; renal function returned to normal, diarrhea ceased, skin improved and confuεion reεolved. Four dayε after the antibody waε stopped the symptoms recurred and the patient died despite the initiation of a second course of antibody. LO-CD2a thus could be uεed to reverse ongoing immune responses to foreign tissues (allogeneic and xenogeneic, since it inhibits the xeno MLR aε well as the allo MLR) . The antibody would be given by i.v. infusion once or twice a day for 10 days to 14 days. It may also be used prophylactically to prevent activation of T cells aε part of the induction protocol immediately following organ transplantation. Although the preεent invention, in a preferred embodiment, is directed to inhibition of graft rejection, it is to be understood that the scope of the invention is not limited thereto and is generally useful for the inhibition of T-cell activation for any and all purposes. Example 6 Construction and Expression of Chimeric Antibody A. Cloning and Sequencing of V H and V L of LO-CD2a Total RNA was isolated from the cell line LO-CD2a (ATCC HB 11423) according to the method of Chergwin (Biochemiεtry. 18:5294, 1979). mRNA waε then prepared using The oligotex-dT mRNA kit (Qiagen, Chatsworth, CA) . Approximately 200-300 ng mRNA waε reverse transcribed using the RNA-PCR kit from Perkin-Elmer Cetuε (Norwalk, CT) . The reaction was carried out at 42°C for 1 hour. Oligonecleotide primers required for amplification of V H and V L genes were chosen using the following references: 1) Sequences of Proteins of Immunological Interest, Kabat et al., Sth ed., 1991, 2) Orlandi et al. , Proc. Nat'1. Acad. Sci., (USA) 86:3833-3837 (1989). V, ___, sense Sma 1 #1 2 3 4 5 6 7 8 5' 3' AA CCC GGG GAC ATT CAG CTG ACC CAG TCT CAA C L antisβnβe Sal 1 #115 114 113 112 111 110 109 5' 3' CA GTC GAC TAC AGT TGG TGC AGC ATC AGC V H sense Sma 1 #1 2 3 4 5 6 7 8 5 ' 3 ' AA CCC GGG GAG GTC CAG CTG CAG CAG TCT GG CH, antisense Sal 1 #124 123 122 121 120 119 5' 3' AAG TCG ACC CAG TGG ATA GAC CGA TGG The numbers refer to amino acid residueε, as shown in Kabat, et al. , 1991. Polymerase chain reactions (PCR) were carried out in a Perkin-Elmer DNA Thermal Cycler 480 uεing the following conditionε: 5 inuteε at 94°C, 30 cycles consiεting of 1 minute at 94°C, 2 minutes at 60°C, and 2 minutes at 72°C. This was followed by 5 minutes at 72°C. DNA fragments were gel purified from 1% agarose uεing the Qiaex gel extraction kit (Qiagen, Chatsworth, CA) . The fragments were then blunt-ended according to the method of Kanungo and Pandey, BioTechniques. 14:912-913 (1993) and ligated into the Sma I site of Bluescript KSII + (Stratagene, La Jolla, CA) . Multiple clones were εequenced by the dideoxy chain termination method uεing the Sequenaεe ™ T7 Polymerase Kit (U.S. Biochemical, Cleveland, OH). Due to the potential error rate inherent in PCR,at leaεt three εeparate reactionε were performed. The most commonly obεerved sequences for LO-CD2a V L and V H genes are shown in Figures 29a and b and 30a and b, wherein Figure 29a shows the nucleotide and amino acid sequences of the LO-CD2a V L chain including the leader sequence from MRC vector hcmv-vllys-kr-neo and Figure 29b showε the nucleotide and amino acid sequenceε of the L0-CD2a V L chain including the leader εequence from the Lo- CD2a gene; Figure 30a shows the nucleotide and amino acid sequences of the LO-CD2a V H chain including the leader sequences from MRC vector hcmv-Vh-Lys-gamma1-neo; and Figure 30b shows the nucleotide and amino acid sequences of the LO-CD2a V H chain including the leader sequences from the LO-CD2a gene. B. Insertion Into Vectors for Transient Expression Two vectors were licensed from The Medical Research Council (MRC) in London for expression of chimeric light and heavy chains of L0-CD2a respectively. The 9.2kb light chain vector (hcmv-vllys-kr-neo) contains the genomic clone of the human kappa constant region and humanized V L domain of anti- lysozyme aε a Hind III-Bam HI fragment. The 8.6kb heavy chain vector (hcmv-VhLys-gamma1-neo) contains the genomic clone of human I constant region and the reshaped V H domain of anti- lyεozyme as a Hind Ill-Bam HI fragment. These vectorε are more fully described in Maeda, et al.. Hum. Antibod. Hybridomas2:124- 134, (1991). Since DNA fragments containing the native signal peptides were unavailable, the V regions of LO-CD2a were cloned behind the signals already present in the MRC vectorε. The light chain V region with signal fragment was constructed from two fragments, each derived from a εeparate PCR reaction as follows: Reaction 1: The DNA template waε the MRC light chain vector. The fragment amplified contained the signal peptide plus a portion of framework (FR)1. The two oligonucleotides used were: Hind III 5'VLlvBsiq (sense) : 5' CCGCAAGCTTCATGGATGGAG 3' Tthlll 3'V lvBsiα (antisense): 5'GCTGCTTGGGGACTGGGTCAGCTGGAT 3' The antisense primer contained the FR 1 sequence of LO- Cd2a, not that found in the MRC vector for anti-lyεozyme. The PCR reaction produced a 0.15Kb Hind III -Tth III fragment. Reaction 2: The DNA template waε the LO-CD2a V L clone in Blueεcript. The fragment amplified included LO-CD2a FR 1 (from the Tth III site) to the end of FR 4. The 3' untranslated region found in the MRC light chain vector waε added to the 3' end of LO-CD2a uεing the antisense oligonucleotide. The 2 oligonucleotides used were: 5' V L LO-CD2a (senεe): Tth in 5'ATTCAGCTGACCCAGTCTCCA 3' 3'VL LQ-CD2a (antisense): BamHI 5'GATCGGATCCACCTGAGGAAGCAAAGTTTAAATTCTACTCAC GTTTCAGTTCCAGCTT 3' This reaction yielded a 0.35Kb Tthlll-Bam HI fragment. Both PCR products were gel purified using Qiaex and restricted with the appropriate enzymes. The Hind III - Tth III fragment plus the Tth III - Bam HI fragment then were ligated between the Hind III and Bam HI sites of Bluescript in a 3-way ligation. This construct, containing the entire V L region of LO-CD2a plus the MRC signal peptide was then sequenced. The heavy chain LO-CD2a V region construct contains the MRC signal sequence at its 5' end and the long 3' untranslated region, also derived from the MRC H chain vector, at its 3' end. The final construct was made from 3 separate PCR reactions aε follows: Reaction 1: The DNA template was the MRC H chain vector. Since V L and V H genes of anti-lyεozyme uεe the εame signal, the εenεe primer was the same as that used for the LO-CD2a V L construct, i.e., 5' V. lyssiσ. The antisense primer was 3'V H lysεiσ: st 1 5'TCTCGTGCAGTGGGACCTCGGAGTGGACACC3' This reaction produced a 0.16Kb Hind III - Pst I fragment containing the MRC signal plus a portion of FR 1 of LO-CD2a. The fragment was gel purified, restricted, and ligated into Hind III - Pst I cut Bluescript for sequencing. Reaction 2: The DNA template was the LO-CD2a V H region in Bluescript. This reaction yielded a 0.3Kb Pεt I - Sty I fragment containing most of the V H region. Because there was an internal Pst 1 site in FR 3 of LO-CD2a, the Pεt I - Sty I fragment had to be constructed from 2 PCR reactions aε follows: 2 4 Sty 1 0.1 Kb FR4 The template DNA shown above is clone 82-8, L0-CD2a V H in Bluescript. Reaction A: Yields a 0.2 Kb fragment, using olignucleotides, also refered to as oligoε 1 and 2, aε primerε: Oligo 1 is : 5 ' Pst I B2-B ( sense ) : Pst I 5 'GAGGTCCAGCTGCAGCAGTCT3 ' Oligo 2 is : 3 ' int . Pet I ( antisense ) : 5 ' CGATGTATCAGCTGTCAGTGTGGC3 ' Reaction B : Yields a 0.1 Kb fragment, uεing oligoε 3 and 4 aε primers . Oligo 3 is 5 ' int . Pεt I ( sense) : 5'GCCACACTGACAGCTGATACATCG3' Oligo 4 is 3'Stv I 82-8 (antisense): Stv I 5'CAGAGTGCCTTGGCCCCAGTA3' Oligoε 2 and 3 above contain changeε in nucleotide εequence which remove the internal Pεt I εite without changing the amino acid εequence of LO-CD2a. Aliquots (2-5μl) of the overlapping productε of reactionε A & B above were combined and served aε templates for a third PCR reaction. The oligonucleotide primers for this reaction were numbers 1 and 4 from the previous diagram. The 0.3 Kb product waε gel purified by Qiaex and restricted with Pst I and Sty I. Since the fragment remained intact, the internal Pst 1 εite had been εucceεεfully mutated. Reaction 3: The final V H fragment waε produced using the MRC heavy chain vector as template. This 0.23 Kb Sty I - Bam HI fragment contained a portion of FR4 of Lo-CD2a, and the entire 3' untranslated region from the MRC vector. The primers used were: 5'V H lyε Sty I (sense): Stv I 5'TACTGGGGCCAAGGCACCCTCGTCACA3' 3 ' Vplvs Bam HI ( antisense ) : Bam HI 5 'GATCGGATCCCTTATAAATCTCTGGC3 ' The resulting fragment was gel purified and restricted with Sty I and Bam HI. The Pst I-Sty I and Sty I-Bam HI frag entε were then ligated into Pεt I-Bam HI cut Blueεcript for sequencing. All oligonucleotides were synthesized on an Applied Biosystems synthesizer. All sequencing reactions were carried out uεing The Sequenaεe ™ T7 Polymeraεe Kit (U.S. Biochemical, Cleveland, OH). All PCRε were carried out using the following protocol: 5 min. at 95°C, 35 cycles consisting of 1 min. at 94°C, 1 min. at 50°C, 2 min. at 72°C, a final extension of 5 min. at 72°C. LO-CD2a V L and V H fragments containing the correct sequences were removed from Bluescript and cloned between the Hind III and Bam HI εiteε of the MRC light and heavy chain vectors, respectively. For the H chain, the 5' Hind III - Pst I fragment was first joined to the remainder of the construct (Pstl-Bam HI) in Bluescript; the entire Hind III - Bam HI fragment waε then cloned into the MRC vector. C. N-Ter inal Amino Acid Sequencing of V H and V L N-terminal amino acid εequence analysis was performed by Harvard Microchemistry Laboratory in Cambridge, MA on samples of LO-CD2a heavy and light chains in order to confirm the sequenceε obtained uεing RNA-PCR. The sampleε were prepared aε follows: -200μg of LO-CD2a waε applied acroεs a 12% SDS polyacrylamide gel run in the presence of B-mercaptoethanol. Following electrophoresiε, the protein was transferred to a PVDF membrane using a Western transfer apparatuε. The membrane was stained briefly with Ponceau S, destained in 1% acetic acid, and the light and heavy chain bands were dried under vacuum and sent for amino acid analyses and N-terminal sequencing. The amino acid sequence of the first 20 residues of LO- CD2a V H agreed completely with the cloned sequence; however, the sequence of V L indicated that residueε 2, 3 and 7 in FRl were different than thoεe encoded by the cloned geneε. These differenceε all reεide in the PCR primer uεed for cloning purposes, based on a best guesε εequence obtained from the previouεly cited literature. D. DNA Sequence Confirmation of N-Terminal Amino Acid Sequence and its Correction In order to correct this sequence and simultaneously clone the native signal peptides of both V L and V H of LO-CD2a, RACE-PCR was employed was employed (Rapid Amplification of cDNA Ends): mRNA from LO-CD2a cellε waε reverse transcribed and the resulting cDNA waε G-tailed at its 3' end using terminal tranεferase in the presence of dGTP. The cDNA was then amplified using a specific 3' oligonucleotide and a 5' oligonucleotide complementary to the G-tail. To simplify subcloning, a suitable reεtriction εite waε added to the 5' end of each oligonucleotide. The oligonucleotideε uεed for preparation of cDNA were aε followε: Bam HI Not I Sal I 3' oligo Vk(VKA) TTGGATCCGCGGCCGCGTCGACTACAGTTGGTGCAGCATCAGC Bam HI Not 1 Sal 1 3' oligo Vh(CHA) ATGGATCCGCGGCCGCGTCGACCCAGTGGATAGACCGATGG The oligonucleotides for RACE-PCR were as follows: Xho I 5' Primer (TV1): 5' CCA TGG CCT CGA GGG CCC CCC CCC CCC CCC C 3' Stu I 3' oligo Vh (BHA) 5* CCT GTT TAG GCC TCT GCT TCA CCC AGT AC 3' Sph I 3' oligo Vk (BKA) 5' GGA TAA TGG GTA AAT TGC ATG CAG TAA TA 3' RACE-PCR reactionε were carried out uεing the following protocol: 5 min. at 94°C, 40 cycleε including 30 sec. at 94°C, 30 sec. at 50°C, and 50 sec. at 72°C, followed by a 5 min. extension at 72°C. PCR products obtained for LO-CD2a V L and V H were gel extracted uεing Qiaex. The V H fragment waε restricted with Xho I and Stu I and ligated into Xho I - Sma I cut Bluescript. The V L fragment was blunt-ended and ligated into Sma I cut Blueεcript. A number of clones were sequenced for both light and heavy chain V regions and the signal sequences were identified. Since signal sequences found in immunoglobulin genes generally have introns, these may be important for expression. Genomic clones containing the V L and V H leader sequences were identified as well. Genomic DNA waε prepared aε followε: 4 x 10 7 LO-CD2a cellε were εpun down, waεhed in cold PBS, εpun down, and washed with PBS again. Cellε were resuεpended in 0.4ml digeεtion buffer (with freεhly added proteinase K) . This mixture was incubated with shaking at 50°C for 12-15 hours, extracted with an equal volume of phenol/chloroform/iεoamyl alcohol, and εpun at 1700xg. The aqueouε phase waε tranεferred to a clean tube and 1/2 volume of 7.5 M ammonium acetate and 2 volumes of 95% ethanol were added. The DNA was pelleted by εpinning 2 minutes, 1700xg. The pellet waε washed with 70% ethanol and air dried. The pellet waε resuspended in 80 ml TE, pH 8.0. Using genomic DNA obtained from the cell line LO-CD2a aε a template, the following oligonucleotides were designated in order to amplify the genomic leader sequenceε of both V L and V H as well as portions of the framework regions ending at unique restriction sites (Sph I for V L , Pst I for V H ) .• LVH #430 TGCAAGCTTCATGATGAGTCCTGTCCAGTC Leader V L sense/Hind III LVL #429 AGTAAGCTTCATGAAATGCAGGTGOATC Leader V H sense/Hind III PVHA # 428 GGαAGArrGCTGC A GC T GGAC TT c V H antisense/Pst I PCR reactionε were carried out aε follows: 100 ng genomic DNA from LO-CD2a cellε, 200 pmol each of oligoε LVL and BKA (for V L fragment) or 200 pmol each of LVH and PVHA (for V H fragment), 100 μl 1mm dNTPε, 10 μl 10 x Pfu buffer. 1 ml (2.5 unitε) pfu DNA polymerase (Stratagene, La Jolla, CA) deionized water to 100 μl. Pfu was used because of its greater accuracy than Taq polymeraεe. The reaction conditionε were aε followε: 5 min. 94°C, 5 min. 50°C, 35 cycleε of 1 min. 94°C, 1 min. 50°C, 1 min. 72°C, followed by 5 min. at 72°C. The PCR products were gel purified, restricted and ligated into Bluescript for sequencing. Once clones containing the correct εequence were identified, Blueεcript vectorε containing theεe clones were cut with Hind III and Sph I (V L ) or Hind III and Pεt I (V H ) and the fragments were gel isolated. The 0.75 Kb Hind Ill-Sph I fragment was then ligated into Bluescript containing the original LO-CD2a V L construct from which the Hind III - Sph I fragment had been removed. The new construct contained the native LO-CD2a signal plus intron and a corrected FRl εequence (in agreement with the N-terminal sequence). The 0.16 Kb Hind III - Pst I fragment waε ligated into Bluescript containing the original LO-CD2a V H construct from which the Hind III - Pst I fragment had been removed. The new construct contained the native εignal + intron. The newly constructed V L and V H fragments were then removed from Bluescript by digestion with Hind III and Bam HI and cloned into the MRC light and heavy chain vectorε, reεpectively, for expreεεion in COS cellε. E. Transient expression in COS cells. COS 7 cells were obtained rom the ATCC and were grown in Dulbecco'ε Minimal Essential Medium (DMEM) with 10% fetal bovine serum (FBS). Optimal transfection was achieved at approximately 50% confluency of adherent cells. In preparation for transfection, plasmid DNA was added to DMEM containing NuSerum and DEAE-Dextran/chloroquine diphosphate. COS cell medium was removed, the DNA mixture waε added and the cellε incubated for 3 hourε at 37°C. Thiε medium then waε removed, and 10% DMSO in PBS was added to the cells for 2 minutes and then removed. DMEM with 10% FBS waε added to the cells. After overnight incubation, the medium waε replaced and the cellε were incubated for 2 days at 37°C. Supernatants were collected for assay by ELISA for the secretion of chimeric antibody. F. Detection of secreted chimeric by ELISA. Secretion of chimeric antibody waε confirmed by assay of supernatants from the tranεfected COS cellε in an ELISA deεigned to detect the presence of human antibody (or a portion thereof). Goat anti-human IgG (H+L) was diluted in phosphate buffered εaline (PBS) to a concentration of 5 μg/ml and bound to the wells of ELISA microtiter plates by overnight incubation at 4°C. Plates were washed 3 times using an ELISA plate washer. Remaining free sites were blocked by the addition of 200 μl PBS containing 1% bovine serum albumin (PBS-BSA) for 1/2 hr. at room temperature. Two-fold dilutions were prepared in PBS-BSA of the supernatants and of a positive control reference standard (purified human igGlk) . Media alone and/or PBS-BSA alone constituted negative controls. Antibody dilutions and controls were added to the wellε and incubated at room temperature for 1 1/2 hourε. Plateε were then waεhed 3 timeε with a plate washer in PBS containing 0.05% Tween20. The appropriate dilution of a goat anti-human IgG (gamma chain specific)-horseradish peroxidase (HRP) conjugated antibody or goat anti-human kappa light chain -HRP conjugated antibody was added to each well and incubated at room temperature for 1 hour. Plates were washed with PBS-Tween20 as described above, after which the developing substrate, (ABTS) containing hydrogen peroxide, was added. Bound antibody was detected by reading abεorbance at a wavelength of 405 nm. 6. Binding specificity of secreted chimeric antibody. Binding specificity of the chimeric was evaluated by flow cytometric analysis of antibody binding to the CD2-expresεing mutant Jurkat cell line JRT3-T3-5. The binding profile of the chimeric antibody (human IgGl) waε compared with those of the native rat antibody (IgG2b) and the iεotype-matched control MABε (human IgGl and rat IgG2b) which exhibit irrelevant (non-CD-2) binding specificities. Preparation of JRT3-T3-5 (Jurkat ) cell line . The Jurkat cell line was obtained from the ATCC ( ) and was propagated in D-MEM containing 10% fetal bovine serum (FBS), 10% amino acid supplement (NCTC), and 6 mM L-glutamine (complete medium). The cellε were maintained at 37°C with 10% C0 2 and were paεεaged three times per week at a ratio of 1:4 (the cell concentration at passage being approximately 3 x 10 6 /ml). Jurkat cells were harvested, centrifuged to remove spent medium, and washed in DMEM. The cells were then resuεpended in phosphate buffered saline (PBS) with 0.1% sodium azide (NaAz), and an aliquot waε removed for cell quantification. The number of viable cellε was determined by trypan blue exclusion. Indirect staining of Jurkat cellε . Cell surface staining was carried out in a 96 well U-bottom icrotiter plate. Approximately 6 x 10 5 cells in a volume of 90 μl were distributed into each well of the microtiter plate. Dilutions of the antibodies to be tested were prepared in PBS with 0.1% NaAz and distributed into the appropriate wells in a volume of 10 μl. Cells were incubated with antibody for 15 minutes at room temperature, after which the cells were washed 3 times by adding PBS with 0.1% NaAz to each well and by centrifuging for 2 minutes at 1900 rpm (Sorvall RT6000D) . Resuεpension of cellε waε accomplished by gently tapping the plates. Ten ul aliquots of the appropriate fluorescein-isothiocyanate (FITC)-conjugated εecondary antibody (anti-human Ig or anti-rat Ig) was added to the appropriate wells and incubated at room temperature for 15 minuteε in the dark. Plates were washed 3 times in PBS with 0.1% NaAz aε described above. Stained cells were fixed by the addition of 200 μl of 0.5% paraformaldehyde in PBS and were stored at 4°C (up to 1 week). Flow cytometric analysis of stained Jurkat cells. Stained cells were transferred to 12 x 17 mm polystyrene tubeε for acquisition of data using a Becton-Dickinεon FACScan. Data acquisition and analysis were carried out using LYSIS-II software. CD2- expressing Jurkat cells were incubated with the LO CD2a (rat IgG2b)MAB, the chimeric version of LO-CD2a (human IgGl), and the correεponding iεotype matched controls. Bound antibody waε detected uεing the appropriate FITC-conjugated εecondary antibody according to the protocol described above. Analysiε εhows εimilar binding patternε of the native rat LO CD2a and the chimeric human-rat LO-CD2a. Numerouε modificationε and variationε of the preεent invention are possible in light of the above teachings and, therefore, within the scope of the appended claimε, the invention may be practiced otherwiεe than aε particularly deεcribed.

Claims: CLAIMS : 1. A compound which binds to the same epitope or a portion thereof on human lymphocytes as the monoclonal antibody produced by the cell line deposited aε ATCC HB 11423. 2. A compound as in Claim 1, wherein said compound is a monoclonal antibody or fragment thereof. 3. A compound aε in Claim 1, wherein said compound is a monoclonal antibody identical to the monoclonal antibody produced by said deposited cell line. 4. The compound of Claim 1 wherein said compound is a monoclonal antibody having the same CDR aε the antibody produced by said deposited cell line. 5. The compound of Claim 1 wherein εaid compound is a humanized form of the monoclonal antibody produced by said deposited cell line. 6. The compound of Claim 1 wherein said epitope is a CD2 epitope of CD2 poεitive human T-cellε. 7. The compound of Claim 1 wherein the epitope is a conformational epitope. 8. The compound of Claim 1 wherein said compound is a monoclonal antibody and said antibody is further characterized by binding to at least a portion of human CD2 + NK cellε. 9. A process for inhibiting an immune responεe in a human patient, comprising: treating a patient by administering an effective amount of the antibody of Claim 2. 10. A procesε aε in Claim 9 wherein said immune response is mediated by T cell activation and proliferation. 11. A procesε aε in Claim 10, wherein said T cell activation and proliferation results from graft transplantation. 12. The procesε aε in Claim 11, wherein said graft transplantation is allograft transplantation. 13. A process as in Claim 12, wherein said graft transplantation is xenograft transplantation. 14. A procesε aε in Claim 10, wherein said T cell activation and proliferation resultε from an autoimmune diseaεe. 15. A procesε aε in Claim 10, wherein the antibody iε contacted with blood of the patient in vivo. 16. A process as in Claim 15, wherein εaid antibody iε contacted with the patient's blood in vivo by intravenous administration. 17. A proceεε aε in Claim 10, wherein said Antibody is contacted with the donor graft prior to transplantation. 18. A process as in Claim 9, wherein said immune response is mediated by natural killer cells. 19. A process as in Claim 18, wherein the antibody iε contacted with blood of the patient in vivo. 20. A procesε as in Claim 19, wherein said antibody is contacted with the patient'ε blood in vivo by intravenouε adminiεtration. 21. A proceεs as in Claim 18, wherein εaid antibody iε contacted with the donor graft prior to tranεplantation. 22. A process as in Claim 18, wherein said immune response reεultε in graft versus host disease. 23. A process as in Claim 18, wherein said immune response results in graft rejection. 24. A process as in Claim 23, wherein εaid graft rejection iε from allograft transplantation. 25. A procesε aε in Claim 23, wherein said graft rejection iε from xenograft tranεplantation. 26. A process for inhibiting the rejection of a graft in a human patient comprising: treating a human patient to inhibit rejection of a graft with a compound, which binds to at least a portion of the same epitope on human lymphocytes as the monoclonal antibody produced by the cell line deposited as ATCC HB 11423, in an amount effective to inhibit rejection. 27. A process as in Claim 26, wherein the compound iε a LO-CD2a antibody. 28. A process as in Claim 26, wherein the graft is an organ. 29. The compound of Claim 5 wherein said antibody includes a V L chain having an amino acid sequence as shown in Figure 29A. 30. The compound of Claim 5 wherein said antibody includes a V H chain having an amino acid sequence as shown in Figure 30A. 31. The compound of Claim 2 wherein εaid antibody includeε a V L chain having an amino acid εequence aε shown in Figure 29B. 32. The compound of Claim 2 wherein said antibody includeε a V H chain having an amino acid εequence aε shown in Figure 3OB.

Inventors: BAZIN HERVE, LATINNE DOMINIQUE, BAZIN, HERVE, LATINNE, DOMINIQUE
Applicants: BAZIN HERVE, LATINNE DOMINIQUE, UNIV LOUVAIN, BAZIN, HERVE, LATINNE, DOMINIQUE, UNIVERSITE CATHOLIQUE DE LOUVAIN
Application year: 1994
Main classification: C12N15
IPC codes: A61K38, A61K39, A61P37, C07K16, C12N15, C12P21, C12R1